Project Summary Nicotinamide adenine dinucleotide (NAD+) is the substrate for sirtuins and polyADP-ribose polymerases (PARPs), linking it to gene expression and genomic stability. These enzymatic activities also connect NAD+ to such aging-related conditions as diabetes and muscle weakness. NAD+ alterations also figure prominently in the relationship between calorie restriction (CR) and disease prevention, but the exact nature of this link remains unknown. One model is that CR elevates intracellular NAD+, which then controls activity of sirtuins that regulate fuel utilization and expression of nutrient-responsive genes. PARP activation can also influence cellular energy homeostasis by depleting NAD+, ultimately leading to cell death. It is important to remember that the contribution of NAD+ to sirtuins and PARPs depends entirely upon the free NAD+ concentration and not on redox, that is, the NAD+/NADH ratio. Although measuring the NAD+/NADH ratio is straightforward, monitoring NAD+ is not?our development of an NAD+ biosensor has provided the first glimpses into NAD+ regulation within subcellular compartments of intact cells. This is an important advance because previous studies could not distinguish free from bound NAD+ or monitor differences in NAD+ regulation across compartments. With this novel sensor in hand, we will determine how NAD+ levels in pancreatic beta cells and skeletal muscle are regulated during aging and whether age-related changes can be prevented by CR or augmentation of NAD+ production. Although we have gained significant insights into NAD+ biology using the current sensor, it would be of great value to extend our studies into intact animals. Thus, our first goal is to develop a conditional transgenic mouse line expressing the NAD+ biosensor. We have already made significant progress optimizing our sensor for in vivo dynamic measurements and describe strategies for increasing its sensitivity and dynamic range further. We will then test whether aging decreases, and CR or NAD+ precursor administration increases, NAD+ levels in pancreatic beta cells and skeletal muscle cells by generating tissue- specific sensor strains capable of monitoring NAD+ levels in the nucleus, cytoplasm, and mitochondria. NAD+ depletion is thought to mediate age-related decreases in insulin secretion. Similarly, age-dependent NAD+ decreases have been proposed to underlie muscle weakness and impairments in muscle regenerative capacity. Our biosensor provides an unprecedented opportunity to examine the effect of aging on NAD+ levels, the contribution of NAD+ to age-related disorders, and the efficacy of several proposed approaches to ameliorating these conditions.